MicroRNAs (miRNAs) are short oligonucleotides that regulate more than one half of protein coding genes' expression in human. MicroRNAs have been associated with common human diseases such cancer, immune disorders, viral infections and neurodegeneration. Since elevated miRNA levels are often linked to various disease states, the biogenesis of miRNA is a tightly regulated process. Dicer mediated maturation of precursor miRNA (pre-miRNA) in the cytoplasm is a critical step in miRNA biogenesis where RNase enzyme Dicer recognizes and internally cleaves the pre-miRNA stem-loop to produce the mature miRNA. The current dogma is that all pre-miRNAs adopt a stem-loop structure, which is required for the accurate maturation by Dicer. Thus, besides the canonical stem-loop structure, no other alternative secondary structures have been actively considered for the pre-miRNAs. However, after comprehensive analysis of all known human pre-miRNA and mature miRNA sequences, we identified 298 and 179 sequences respectively which have the propensity to form a stable G-quadruplex (GQ) as an alternative RNA secondary structure. In this proposal, we intend to investigate in detail the role of GQ structure in miRNA biogenesis and the ability of small molecules to control the pre-miRNA maturation by targeting the GQ structures. Using a clinically important miRNA (pre-miRNA 92b) which is overexpressed in various diseases, we showed the structural coexistence of GQ and the canonical stem-loop structures of pre-miRNA under the physiologically relevant K+ concentration. Additionally, our preliminary data show that the GQ formation inhibits the Dicer mediated maturation of human pre-miRNA 92b. Given these findings, we hypothesize that GQ structures play a crucial role in miRNA biogenesis at both pre-miRNA maturation and mature duplex miRNA strand separation. We also hypothesize that small molecule binding to the GQs in pre-miRNA can modulate its maturation. These hypotheses will be investigated under the specific aims: (I) to determine the effect of the GQ on Dicer mediated pre-miRNA maturation using human pre-miRNA 92b, (II) to delineate whether the GQ formation in the mature miRNAs causes the structural unwinding of the miRNA duplex; and (III) to determine if small molecule targeted to non-canonical structure in pre-miRNA can modulate its maturation by Dicer in vitro and in human cells. Completion of these specific aims will reveal the importance of alternative secondary structures in miRNA biogenesis, which in turn will lead to better understanding of how miRNA levels control various diseases. The targeting of GQs with small molecule will be a new way to control the endogenous miRNA biogenesis, which can be used for development of novel therapeutics. This comprehensive study will not only add a new dimension to our current understanding of the RNA structural role in miRNA maturation, but also open up a new therapeutic strategy to treat various diseases that are linked to overexpression of G-rich miRNAs.